Protein d - an igd-binding protein of haemophilus influenzae

ABSTRACT

A novel surface exposed protein of  Haemophilus influenzae  or related  Haemophilus  species is described. The protein named protein D is an Ig receptor for human IgD and has an apparent molecular weight of 42,000. Protein D can be detected in all of 116 encapsulated and non-encapsulated isolates of  H. influenzae  studied. The protein from all strains shows in addition to the same apparent molecular weight immunogenic similarities since protein D from all strains interacts with three different mouse monoclonal antibodies and monoclonal human IgD. A method for purification of protein D is described. Cloning of the protein D gene from  H. influenzae  in  E. coli  is described as well as the nucleotide sequence and the deduced amino acid sequence.

This is a continuation of application Ser. No. 11/521,598, filed Sep.15, 2006, now allowed, which is a continuation of application Ser. No.09/607,933, filed Jun. 30, 2000, now U.S. Pat. No. 7,115,271, which is adivision of application Ser. No. 09/225,443, filed Jan. 6, 1999, nowU.S. Pat. No. 6,139,846, which is a division of application Ser. No.08/936,912, filed Sep. 25, 1997, now U.S. Pat. No. 5,888,517, which is acontinuation of application Ser. No. 08/468,618, filed Jun. 6, 1995, nowabandoned, which is a continuation of application Ser. No. 07/946,499,filed Nov. 9, 1992, now abandoned, which is the National Stage ofInternational Application No. PCT/SE91/00129, filed Feb. 21, 1991, allof which are incorporated herein by reference.

The present invention is related to a surface exposed protein namedprotein D which is conserved in many strains of Haemophilus influenzaeor related Haemophilus species. Protein D is an Ig receptor for humanIgD.

Several immunoglobulin (Ig) binding bacterial cell wall proteins havebeen isolated and/or cloned during the last two decades. The bestcharacterized of these are protein A of Staphylococcus aureus andprotein G of group G beta-hemolytic streptococci. The classicalFc-binding capacity of protein A involves IgG from humans and severalmammalian species but the binding is restricted to human IgG subclasses1, 2 and 4. Also other human classes of Ig (G, A, M, E) have been shownto bind to protein A, a reactivity that has been designed thealternative Ig binding which is mediated by Fab structures andcharacterized by a variable occurrence in the different Ig classes.

Protein G of group G streptococci binds all human IgG subclasses and hasalso a wider binding spectrum for animal IgG than protein A. On the IgGmolecule the Fc part is mainly responsible for the interaction withprotein G although a low degree of interaction was also recorded for Fabfragments. IgM, IgA and IgD, however, show no binding to protein G. Bothprotein A and protein G have acquired many applications forimmunoglobulin separation and detection. (EP 0 200 909, EP 0 131 142, WO87/05631, U.S. Pat. No. 3,800,798, U.S. Pat. No. 3,995,018.)

Certain strains of group A streptococci are also known to produce anIgG-binding protein which has been purified or cloned. The Ig-bindingprotein from group A streptococci is relatively specific for human IgG.Information about bacterial molecules that selectively bind IgA and IgMis more limited. However, IgA-binding proteins have been isolated fromboth group A and group B streptococci, two frequent human pathogens. TheIgA receptor of group A streptococci has been named protein Arp. Certainstrains of the anaerobic bacterium Clostridium perfringenspreferentially bind IgM but also IgA and IgG. This binding is due to acell surface protein (protein P). Recently a bacterial protein, proteinL, with unique binding properties for L-chains was isolated fromPeptococcus magnus. Protein L has been shown to bind IgG, IgA and IgMfrom human and several mammalian species. Among gram-negative bacteria,Ig receptors have been reported among veterinary pathogens. Brucellaabortus binds bovine IgM and Taylorella equigenitalis, a venerealpathogen of horses, binds equine IgG. Recently Haemophilus somnus wasreported to bind bovine IgG.

A decade ago Haemophilus influenzae and Moraxella (Branhamella)catarrhalis were shown to have a high binding capacity for human IgD(Forsgren A. and Grubb A, J. Immunol. 122:1468, 1979).

The present invention describes the solubilization and purification of aH. influenzae surface protein responsible for the interaction with IgD.It also describes the cloning, expression and nucleotide sequence of theIgD-binding protein gene of the H. influenzae in Escherichia coli. Inaddition it describes the Ig-binding properties of this molecule, namedprotein D, which were found to be different compared with previouslyisolated Ig-binding proteins. Protein D was found only to interact withIgD and not with other human immunoglobulin classes. Thus, protein Dcould be an important tool for studies, separation and detection of IgDin a way similar to the way in which protein A and protein G previouslyhave been used for IgG. Protein D could also be a valuable tool aloneand in combination with other molecules (for example proteins andpolysaccharides) in the stimulation of the immune system through aninteraction with B-lymphocytes. Protein D is not identical with anypreviously described protein from H. influenzae.

H. influenzae is a common human parasite and pathogen which colonizesthe mucosa of the upper respiratory tract and causes disease by localspread or invasion. An important distinguishing feature between H.influenzae isolates is whether or not they are encapsulated.Encapsulated H. influenzae type b is a primary cause of bacterialmeningitis and other invasive infections in children under 4 years ofage in Europe and the United States. Non-encapsulated (non-typable) H.influenzae rarely cause invasive infection in healthy children andadults but are a frequent cause of otitis media in children and havebeen implicated as a cause of sinusitis in both adults and children. H.influenzae are also commonly isolated in purulent secretions of patientswith cystic fibrosis and chronic bronchitis and have recently beenrecognized as an important cause of pneumonia.

A vaccine composed of purified type b capsular polysaccharide has proveneffective against H. influenzae type b disease in children of 2 to 5years of age. However, since children under two years of age respondpoorly to this vaccine, conjugate vaccines with enhanced immunogenicityhave been developed by covalently bonding the capsular polysaccharide tocertain proteins. However; the polysaccharide vaccines; non-conjugatedand conjugated, are of no value against nontypable H. influenzaedisease. Hence, other cell surface components and in particular outermembrane proteins (OMPs) have been looked at as potential vaccinecandidates both against type b and nontypable H. influenzae. (EP 0 281673, EP 0 320 289.)

The outer membrane of H. influenzae is typical of gram-negative bacteriaand consists of phospholipids, lipopolysaccharide (LPS), and about 24proteins. Four different Haemophilus OMPs have been shown to be targetsfor antibodies protective against experimental Haemophilus disease.These include the P1 heat-modifiable major outer membrane protein, theP2 porin protein, the P6 lipoprotein and a surface protein with anapparent molecular weight of 98,000 (98 K protein). Of these at leastantibodies to P2 have been shown not to protect against challenge withheterologous Haemophilus strains. (Loeb, M. R. Infect. Immun. 55:2612,1987; Munson Jr. S. et al J. Clin. Invest. 72:677, 1983; Munson Jr. R.S. and Granoff, D. M. Infect. Immun. 49:544, 1985 and Kimura, A. et al.,Infect. Immun. 194:495, 1985).

Analysis of nontypable H. influenzae has shown that there are markeddifferences in OMP composition among strains (See e.g. Murphy et al. “Asubtyping system for nontypable Haemophilus influenzae based on outermembrane proteins” J Infect Dis 147:838, 1983; Barenkamp et al. “Outermembrane protein and biotype analysis of pathogenic nontypableHaemophilus influenzae” Infect Immun 30:709, 1983).

If a surface exposed antigen (immunogen) which is conserved in allstrains of H. influenzae could be found it would be an important tool indeveloping a method of identifying H. influenzae in clinical specimensas well as a vaccine against H. influenzae. The present invention showsthat protein D with an identical apparent molecular weight (42,000),reacting with three different monoclonal antibodies and human IgD, wasfound in all 116H. influenzae strains (encapsulated and nonencapsulated)studied, as well as in two other related Haemophilus species, namely H.haemolyticus and H. aegypticus.

Thus, according to the invention there is provided a surface exposedprotein, which is conserved in many strains of Haemophilus influenzae orrelated Haemophilus species, having an apparent molecular weight of42,000 and a capacity of binding human IgD. The invention also comprisesnaturally occurring or artificially modified variants of said protein,and also immunogenic or IgD-binding portions of said protein andvariants. The protein is named protein D and has the amino acid sequencedepicted in FIG. 9 (SEQ ID NO: 3).

There is also provided a plasmid or phage containing a genetic code forprotein D or the above defined variants or portions.

Further there is provided a non-human host containing the above plasmidor phage and capable of producing said protein or variants, or saidportions thereof. The host is chosen among bacteria, yeasts or plants. Apresently preferred host is E. coli.

In a further aspect the invention provides for a DNA segment comprisinga DNA sequence which codes for protein D, or said variants thereof, orfor said portions. The DNA sequence is shown in FIG. 9 (SEQ ID NO: 1).

In yet another aspect, the invention provides for a recombinant DNAmolecule containing a nucleotide sequence coding for protein D, or saidvariants or portions, which nucleotide sequence could be fused toanother gene.

A plasmid or a phage containing the fused nucleotide defined above couldalso be constructed.

Further such a plasmid or phage could be inserted in a non-human host,such as bacteria, yeasts or plants. At present, E. coli is the preferredhost.

The invention also comprises a fusion protein or polypeptide in whichprotein D, or said variants or portions, could be combined with anotherprotein by the use of a recombinant DNA molecule, defined above.

Furthermore, a fusion product in which protein D, or said variants orportions, is covalently or by any other means bound to a protein,carbohydrate or matrix (such as gold, “Sephadex” particles, polymericsurfaces) could be constructed.

The invention also comprises a vaccine containing protein D, or saidvariants or portions. Other forms of vaccines contain the same protein Dor variants or portions, combined with another vaccine, or combined withan immunogenic portion of another molecule.

There is also provided a hybridoma cell capable of producing amonoclonal antibody to an immunogenic portion of protein D, or ofnaturally occurring or artificially modified variants thereof.

Further there is provided a purified antibody which is specific to animmunogenic portion of protein D or of naturally occurring orartificially modified variants thereof. This antibody is used in amethod of detecting the presence of Haemophilus influenzae or relatedHaemophilus species in a sample by contacting said sample with theantibody in the presence of an indicator.

The invention also comprises a method of detecting the presence ofHaemophilus influenzae or related Haemophilus species in a sample bycontacting said sample with a DNA probe or primer constructed tocorrespond to the nucleic acids which code for protein D, or fornaturally occurring or artificially modified variants thereof, or for animmunogenic or IgD-binding portion of said protein or variants.

Protein D, or said variants or portions, is also use in a method ofdetecting IgD. In such a detecting method the protein may be labelled orbound to a matrix.

Finally, the invention comprises a method of separating IgD usingprotein D, or said variants or portions, optionally bound to a matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of % ¹²⁵I-IgD bound versus serotypes and biotypesshowing that all H. influenzae isolates bound IgD to a high degree.

FIG. 2 is a direct binding assay demonstrating that of the bacteriatested, only H. haemolyticus and H. aegypticus bound radiolabeled IgD.

FIG. 3 depicts stains and electroblots of solubilized proteins.

FIG. 4 depicts electroblots of cell debris which were probed with IgDdemonstrating that Sarcosyl treatment effectively solubilized protein D.

FIG. 5 depicts a reelectrophoresis of purified protein D.

FIGS. 6A, 6B, 6C and 6D are graphs which depict the interaction ofprotein D with human IgD when the proteins were run on a Sephadex G-200column.

FIG. 7 is a dot blot which shows that protein D effectively bound tohighly purified human IgD myeloma proteins.

FIG. 8 is a partial restriction enzyme map for the insert of H.influenzae DNA in pHIJ32.

FIGS. 9 a and 9 b present a DNA sequence (SEQ ID NO: 1) encoding aprotein D amino acid sequence (SEQ ID NO: 3).

FIG. 10 depicts an immunoblotting experiment which analyzed protein Dexpressed in E. coli JM83 carrying pHIC348.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods Bacteria

116H. influenzae strains representing serotypes a-f and nontypable andin addition bacterial strains representing 12 species related to H.influenzae were obtained from different laboratories in Denmark, Swedenand the U.S.A.

Culture Conditions

All strains of Haemophilus, Ekinella and Acinobacillus were grown onchocolate agar. H. ducreyi were grown in microaerophilic atmosphere at37° C. and all other Haemophilus strains in an atmosphere containing 5%CO₂. 30 isolates of H. influenzae were also grown overnight at 37° C. inbrain-heart infusion broth (Difco Lab., Inc. Detroit, Mich.)supplemented with nicotinamide adenine dinucleotide and hemin (SigmaChemical Co. St Louis, Mo.), each at 10 μg/ml.

Immunoglobulins and Proteins

IgD myeloma proteins from four different patients were purified asdescribed (Forsgren, A. and Grubb, A., J. Immunol. 122:1468, 1979).Eight different human IgG myeloma proteins representing all foursubclasses and both L-chain types, three different IgM myeloma proteinsand one IgA myeloma protein were isolated and purified according tostandard methods. Human polyclonal IgG, serum albumin and plasminogenwere purchased from Kabi Vitrum AB, Stockholm, Sweden, and human IgE wasadapted from Pharmacia IgE RIACT kit (Pharmacia Diagnostic AB, Uppsala,Sweden). Bovine serum albumin, human and bovine fibrinogen and humantransferrin were purchased or obtained as a gift.

¹²⁵I-IgD Binding Assay

The binding assay was carried out in plastic tubes. Briefly 4×10⁸bacterial cells in a volume of 100 μl phosphate buffered saline (PBS)with the addition of 5% human serum albumine (HSA) were mixed with 100μl of ¹²⁵I-IgD in the same buffer (radioactivity was adjusted to 7-8×10⁴cpm, i.e approx. 40 ng). After 0.5 h incubation at 37° C., 2 ml ofice-cold PBS (containing 0.1% Tween 20) was added to the tubes.

The suspension was centrifugated at 4,599×g for 15 min and thesupernatant was aspirated. Radioactivity retained in the bacterialpellet was measured in a gamma counter (LKB Wallac Clingamma 1271,Turku, Finland). Residual radioactivity from incubation mixturescontaining no bacteria, i.e. background, was 2.5 percent. Samples werealways tested in triplicates and each experiment was repeated at leasttwice, unless otherwise stated.

Monoclonal Antibodies

Inbred female BALB/c mice (age 8 to 14 weeks) were immunized by anintraperitoneal injection of 25 μg purified protein D (25 μg/50 μl inFreund's complete adjuvant (300 μl) followed by two intraperitonealinjections of protein D (15 μg) in Freund's incomplete adjuvant (300 μl)3 and 7 weeks later. In week 9 the mice were bled from the tails, serumwas separated and tested for anti-protein D activity in an enzyme-linkedimmunosorbent assay (ELISA). The best responding mouse was boosted by anintravenous injection of protein D (2 μg) in 150 μl PBS. One day afterthe last injection, the spleen was excised and spleen cells wereprepared for the production of monoclonal antibodies (De St Groth S F,Scheidegger S J J Immunol Methods 35:1, 1980). After 10 to 14 days (mean12 days) the hybridomas were tested for the production of antibodiesagainst protein D in an enzyme-linked immunosorbent assay (ELISA), andthe hybrids producing the highest titers of antibodies were cloned andexpanded by cultivation in RPMI medium containing 10% fetal bovineserum. Totally 68 clones producing antibodies to protein D wereobtained. Three of the hybridomas were selected for further growth inthe same medium. All cell lines were frozen in the presence of dimethylsulfoxide and 90% fetal bovine serum in liquid nitrogen.

SDS-PAGE and Detection of Protein D on Membranes

SDS-PAGE was, using a modified Laemmli gel, prepared and run accordingto the procedure of Lugtenberg et al., (FEBS Lett 58:254, 1975) using atotal acrylamide concentration of 11%. Samples of crude Sarcosylextracts of H. influenzae and related bacterial species were pretreatedby 5-min boiling in sample buffer consisting of 0.06M of Trishydrochloride (pH 6.8), 2% (w/v) SDS, 1% (v/v) β-ME, 10% glycerol, and0.03% (w/v) bromphenol blue. Electrophoresis was performed at roomtemperature using PROTEIN II vertical slab electrophoresis cells(Bio-Rad Laboratories, Richmond, Calif.) at 40 mA per gel constantcurrent. Staining of proteins in gels was done with comassie brilliantblue in a mixture of methanol, acetic acid and water essentially asdescribed by Weber and Osborn (J. Biol. Chem. 244:4406, 1969). Proteinbands were also transferred to nitrocellulose membranes (Sartorius, WestGermany) by electrophoretic transfer from SDS-polyacrylamide gels.Electrophoretic transfer was carried out in a Trans-Blot Cell (Bio-Rad)at 50 V for 90 min. The electrode buffer was 0.025M Tris, pH 8.3, 0.192Mglycine, and 20% methanol. The membranes were then washed for 1 h atroom temperature in 1.5% ovalbumin-Tris balanced saline (OA-TBS), pH7.4, to saturate additional binding sites.

After several washings with Tris balanced saline (TBS), the membraneswere incubated overnight at room temperature in 1% OA-TBS buffercontaining IgD (20 μg/ml) to detect IgD-binding bands, then washed twicewith TBS. The membranes were then incubated with peroxidase conjugatedgoat anti-human IgD (Fc) (Nordic Immunology, Tiiburg, The Netherlands)for 1-2 hrs at room temperature; after several washings with Tween-TBSthe membranes were developed with 4-chloro-1-napthol and hydrogenperoxide. Protein D was also identified using anti-protein D mousemonoclonal antibodies 16C10, 20G6 and 19B4 at 1:50 dilution in 1%OA-TBS. Protein 1 and 2 of H. influenzae were identified using anti-P2mouse monoclonal 9F5 (Dr. Eric J. Hansen, Dallas, Tex., USA) at a 1:1000dilution and rabbit anti-P1 serum (Dr. Robert S. Munson, St. Louis, Mo.,USA) at a 1:200 dilution.

Solubilization and Purification of Protein D from H. Influenzae

Briefly 3 g of bacteria were suspended in 10 ml of 10 mM HEPES Trisbuffer (pH 7.4) containing 0.01M EDTA and sonicated three times in asonifier (MSE) for 1 min while cooling in an ice bath. Followingsonication Sarcosyl (Sodium Lauryl Sarcosinate) was added to a finalconcentration of 1% (w/v). The suspensions were incubated at noontemperature for 1 h using a shaker and then sonicated again 2×1 min onice and reincubated at room temperature for 30 min. After centrifugationat 12,000 g for 15 min at 4° C. the supernatant was harvested andrecentrifugated at 105,000 g for 1.5 h at 4° C.

Sarcosyl extracts prepared of H. influenzae, strain NT 772 as describedabove were applied to SDS-PAGE. After electrophoresis narrow gel stripswere cut out, protein was transferred to membranes and the IgD-bindingband was detected by Western blot assay using IgD and peroxidaseconjugated goat anti-human IgD as described above (see SDS-PAGE anddetection of protein D on membranes). By comparison with the IgD-bindingband on the membrane (Western blot) the appropriate band in the gelcould be identified and cut out. Electrophoretic elution of theIgD-binding molecules (protein D) was performed and SDS was removed fromthe protein containing solution by precipitation in potassium phosphatebuffer using a method from Susuki and Terrada (Anal. Biochem. 172:259,1988). Potassium phosphate in a final concentration of 20 mM was addedand after incubation at 4° C. overnight the SDS-precipitate was removedby centrifugation at 12,000 g. Thereafter the potassium content wasadjusted to 60 mM and after 4 hrs at 4° C. centrifugation was performedas above. Finally the supernatant was concentrated and extensivedialysis was performed.

Dot Blot Assay

Proteins were applied to nitrocellulose membranes (Schleicher & Schuell,Dessel, West Germany) manually by using a dot blot apparatus (Schleicher& Schuell). After saturation, the membranes were incubated overnight atroom temperature in 1% OA-TBS containing ¹²⁵I-labeled protein probe (5to 10×10⁵ cpm/ml), washed four times with TBS containing 0.02% Tween-20,air dried, and autoradiographed at −70° C. by using Kodak CEA.C X-rayfilms and Kodak X-Omat regular intensifying screen (Eastman Kodak,Rochester, N.Y.).

Amino Acid Sequence Analysis

Automated amino acid sequence analysis was performed with an AppliedBiosystems 470A gas-liquid solid phase sequenator (A) with onlinedetection of the released amino acid phenylthiohydantoin derivatives byApplied Biosystems Model 120A PTH Analyzer.

Bacterial Strains, Plasmids, Bacteriophages and Media Used for Cloningof Protein D

H. influenzae, nontypable strain 772, biotype 2, was isolated from anasopharyngeal swab at the Department of Medical Microbiology, MalmöGeneral Hospital, University of Lund, Sweden. E. coli JM83 were used asrecipient for plasmids pUC18 and pUC19 and derivatives thereof E. coliJM101 and JM103 were used as hosts for M13mp18 arid mp19 bacteriophages.H. influenzae was cultured in brain-heart infusion broth (Difco Lab.,Inc. Detroit, Mich.) supplemented with NAD (nicotine adeninedinucleotide) and hemin (Sigma Chemical Co., St Louis, Mo.), each at 10μg/ml. E. coli strains were grown in L broth or 2×YT media. L agar and2×YT agar contained in addition 1.5 g of agar per litre. L broth and Lagar were, when so indicated, supplemented with ampicillin (Sigma) at100 μg/ml.

DNA Preparations

Chromosomal DNA was prepared from H. influenzae strain 772 by using amodification of the method of Berns and Thomas (J. Mol. Biol. 11:476,1965). After the phenol:chloroform:isoamylalcohol (25:24:1) extractionstep the DNA was ethanol precipitated. The DNA was dissolved in 0.1×SSC(1×SSC:0.15 M NaCl and 0.015 M sodium citrate) and RNase treated for 2 hat 37° C. The RNase was removed with two chloroform:isoamylalcohol(24:1) extractions. The DNA was banded in a CsCl-ethidium bromideequilibrium gradient.

Plasmid DNA and the replicative form of phage M13 from E. coli JM101were obtained by the alkaline lysis procedure followed by furtherpurification in a CsCl-ethidium bromide gradient. In some cases plasmidDNA was prepared using a Quiagen plasmid DNA kit (Diagen GmbHDüsseldorf, FRG).

Single-stranded (ss) DNA from phage M13 clones was prepared from singleplaques (Messing, J. Meth. Enzymol 101C: 20, 1983).

Molecular Cloning of the Protein D Gene

A H. influenzae genomic library was constructed starting from 40 μg ofH. influenzae strain 772 DNA which was partially digested with 1.2 unitsSau3A for 1 h at 37° C. The cleaved DNA was fractionated on a sucrosegradient (Clark-Curtiss, J. E. et al., J. Bacteriol. 161:1093, 1985).Fractions containing DNA fragments of appropriate sizes (2-7kilobasepairs (kbp)) were pooled and the DNA was ligated todephosphorylated BamHI digested pUC18 under standard conditions(Maniatis, T. et al., Molecular cloning: A laboratory manual, 1982). Theligation mixture was transformed into component E. coli JM83 by highvoltage electroporation with a Gene Pulser™/Pulse controller apparatus,both from Bio-Rad Lab. (Richmond, Calif.). The bacteria were plated ontoL agar supplemented with ampicillin and X-gal(5-Bromo-4-chloro-3-indolyl-βD-galactopyranoside).

Colony Immunoassay

For colony immunoblotting, E. coli transformants, cultivated overnighton L agar, were transferred to nitrocellulose filters (Sartorius GmbH,Göttingen, FRG) by covering the agar surfaces with dry filters. Theplates were left for 15 min before the filters were removed and exposedto saturated chloroform vapour for 15 min. Residual protein bindingsites on the filters were blocked by incubating the filters in Trisbalanced saline containing ovalbumine for 30 min (TBS-ova; 50 mMTris-HCl, 154 mM NaCl, 1.5% ova.; pH 7.4). After blocking, the filterswere incubated in turn with (i) culture supernatants containing mousemonoclonal antibodies (MAbs) directed against protein D at a dilution of1:10 in TBS-ova, (ii) horseradish peroxidase conjugated rabbitanti-mouse IgGs (DAKOPATTS A/S., Giostrup, Denmark) in TBS-ova at adilution of 1:2000 in TBS-ova, and (iii) 4-chloro-1-naphthol and H₂O₂.The filters were washed 3×10 min in wash buffer (TBS-0.05% Tween 20)between each step. All incubations were done at room temperature.

Colonies were also checked for IgD binding by incubating other filterswith purified human myeloma IgD:s, rabbit anti-human IgD (δ-chains)(DAKOPATTS), horseradish peroxidase conjugated goat anti-rabbit Ig:s(Bio-Rad Lab.) and 4-chloro-1-naphthol and H₂O₂ as above.

Restriction Endonuclease Analysis and DNA Manipulations

Plasmid and phage DNA were digested with restriction endonucleasesaccording to the manufacturers' instructions (Boehringer Mannheim mbH,Mannheim, FRG, and Beckman Instruments, Inc., England). Restrictionenzyme fragments for subcloning were visualised with low energy UV-lightand excised from 0.7-1.2% agarose gels (Bio-Rad) containing 0.5%ethidium bromide. The DNA bands were extracted with a Geneclean™ kit(BIO 101 Inc., La Jolla, Calif.) as recommended by the supplier.

Ligations were performed with 14 DNA ligase (Boehringer Mannheim) understandard conditions (Maniatis et al., 1982). The ligation mixtures wereused to transform competent E. coli cells.

Progressive deletions of the recombinant plasmid pHIC348 for thesequencing procedure were produced by varying the time of exonucleaseIII digestion of KpnI-BamHI-opened plasmid DNA (Henikoff, S. Gene28:351, 1984). For removal of the resulting single-stranded ends, mungbean nuclease was used. Both nucleases were obtained from BethesdaResearch Laboratories Inc. (Gathersburg, Md.).

Protein D Extraction from E. Coli

Cells of E. coli expressing protein D were grown in L broth supplementedwith ampicillin to early logarithmic phase and then subjected to osmoticshock. After removal of periplasmic fraction the cells were lysed withNaOH (Russel, M. and Model, P., Cell 28:177, 1982) and the cytoplasmicfraction was separated from the membrane fraction by centrifugation. Theperiplasmic and cytoplasmic proteins were precipitated with 5%tri-chloro acetic acid.

DNA Sequencing and Sequence Manipulations.

The nucleotide sequence was determined by direct plasmid sequencing(Chen, E. Y. and Seeburg, P. H. DNA 4:165, 1985) of subclones anddeletion derivatives of plasmid pHIC348 using the chain terminationmethod with α[³⁵S]-dATP (Amersham) and Sequenase™, version 2 (UnitedStates Biochemical Corp., Cleveland, Ohio) following the protocolprovided by the supplier. Part of the sequencing was done onsingle-stranded M13 DNA carrying inserts derived from pHIC348.Autoradiography was performed with Fuji X-ray film.

Results

Distribution of Protein D in Haemophilus influenzae

A total of 116H. influenzae strains obtained from culture collectionsand freshly isolated from nasopharyngeal swabs were selected forIgD-binding experiments. Eleven of the strains were encapsulatedrepresenting serotypes a-f, and 105 strains were non-encapsulated(nontypable). These 105 strains belonged to biotype I (21 strains),biotype II (39 strains), biotype III (14 strains), biotype IV (2strains) and biotype I (5 strains). Of the non-encapsulated strains 31were not biotyped (NBT) but tested for IgD binding.

Approximately 4×10⁸ cfu of H. influenzae bacteria grown on chocolateagar were mixed and incubated with 40 ng of radiolabeled human myelomaIgD. Thereafter a larger volume (2 ml) of PBS containing Tween 20 wasadded, bacteria were spun down and radioactivity of pellets wasmeasured. All H. influenzae isolates bound IgD to a high degree (38-74%)(FIG. 1). There was no difference in IgD-binding capacity betweendifferent serotypes (a-f) of encapsulated H. influenzae. Nor was thereany difference between different biotypes of non-encapsulated strains.30 strains representing different sero- and biotypes were also grown inbrain-heart infusion broth. When those bacteria grown in liquid mediumwere compared with the same bacteria grown on chocolate agar, nodifference in IgD-binding capacity could be detected.

Protein D was solubilized from all 116H. influenzae strains bysonication and Sarcosyl extraction. Subsequently the extracts containingprotein D were subjected to SDS-PAGE. Proteins were stained orelectroblotted onto nitrocellulose membranes and probed with human IgDmyeloma protein and three different mouse monoclonal antibodiesrecognizing protein D. Many protein bands could be detected in allSDS-gels but electrophoresis of extracts from all H. influenzae isolatesgave a protein band with an apparent molecular weight of 42,000 (42kilodaltons). IgD and also all three anti-protein D monoclonalantibodies (16C10, 20G6 and 19B4) bound to the same band afterelectrophoresis of all extracts and subsequent transfer to membranes andblotting.

Bacterial strains of 12 different species taxonomically related to H.influenzae (H. ducreyi, H. paraphrophilus, H. parasuis, H.parainfluenzae, H. haemolyticus, H. parahaemolyticus, H. aphrophilus, H.segnis, H. aegypticus, H. haemoglobinophilus, E. corrodens, A.actinomycetemcomitans) were tested for their capacity to bind ¹²⁵Ilabeled human IgD. In addition crude Sacrosyl extracts from the samebacteria were tested by Western blot analysis with IgD and the threeanti-protein D monoclonal antibodies (MAbs 16C10, 20G6, 19B4).

Of all twelve species tested, only H. haemolyticus (5/5 strains) and H.aegypticus (2/2 strains) bound radiolabeled IgD, 21-28% and 41-48%,respectively, in the direct binding assay (FIG. 2). In Western blotanalysis IgD and all three monoclonal antibodies detected a single bandwith an apparent molecular weight of 42,000 (42 kilodaltons).

None of the 6 strains of H. paraphrophilus, 11H. parainfluenzae, 8 H.aphrophilus, and 3 A. actinomycetemcomitans bound radiolabeled IgD inthe direct binding assay or reacted with IgD in Western blot analysis.However, extracts of all these strains reacted with two or three of themonoclonal antibodies in Western blot analysis showing a single 42kilodaltons protein band. Western blot analysis of three strains of E.corrodens revealed a single high molecular weight band (90 kilodaltons)with MAb 16C10 in all three strains. In an extract of one of thestrains, a single 42 kilodaltons band was detected with the two othermonoclonal antibodies. Two strains of H. ducreyi, H. parasuis (2strains), H. parahaemolyticus (2 strains), H. sengius (2 strains), H.haemoglobinophilus (1 strain) did not bind radiolabeled IgD in thedirect binding assay and Sarcosyl extracts from the same bacteria didnot reveal any protein band detectable by IgD or the three monoclonalantibodies.

Solubilization of Protein D

Three different strains of H. influenzae (two nontypable strains, 772and 3198 and one type B, Minn A.) were grown overnight in broth.Initially attempts were made to solubilize protein D according to a wellestablished method for isolation of H. influenzae outer membraneproteins by sonication, removal of the cell debris by centrifugation andextraction of the supernatant with Sarcosyl followed byultracentrifugation (Barenkamp S J and Munson R S J Infect Dis 143:668,1981). The pellets (cell debris) (d) and supernatants (s) aftersonication as well as the pellets (p) and supernatants (ss) afterSarcosyl-treatment and ultracentrifugation were subjected to SDS-PAGE.Proteins were stained or electroblotted onto Immobilon membranes andprobed with human IgD myeloma protein followed by incubation withperoxidase conjugated anti-human IgD-antibodies and substrate. As shownin FIG. 3 the sonication procedure solubilized proteins includingprotein D effectively. However, IgD-binding molecules (protein D) couldalso be detected in the cell debris, i.e. were not solubilized bysonication. The yield of IgD-binding molecules in the supernatant variedbetween different experiments. FIG. 3 also shows that protein D mostlycould be detected in the Sarcosyl soluble supernatant afterultracentrifugation. In contrast previously described outer membraneproteins of H. influenzae (protein 1 to 6) are readily solubilized bysonication and are considered Sarcosyl insoluble.

To improve the yield of protein D several extraction methods were tried.In subsequent experiments the bacterial cells were sonicated and thewhole cell suspension sonicated and extracted in different detergents(Sarcosyl, NP-40, Triton X-100 and Tween 80). The cell debris wasremoved by centrifugation (12,00 g) and the supernatantultracentrifuged. The thus obtained cell debris (d), supernatants (s)and pellets (p) were analysed by SDS-PAGE, electroblotting ontomembranes and subsequent probing with IgD. As shown in FIG. 4 Sarcosyltreatment effectively solubilized protein D leaving little left in thecell debris and pellet. NP-40, Triton X-100 and Tween-80 solubilizedprotein D less effectively.

Attempts were also made to solubilize protein D from the bacteria withlysozyme and different proteolytic enzymes (papain, pepsin and trypsin)at different concentrations. Of the enzymes only lysozyme solubilizedprotein D (FIG. 4).

Purification of Protein D

Protein D was solubilized by Sarcosyl extraction of whole bacteria asdescribed above and purification was performed by SDS-PAGE of thesupernatant after ultracentrifugation. After electrophoresis narrow gelstrips were cut out, proteins were transferred to membranes and theIgD-binding band (protein D) was detected by Western blot assay. Gelslices containing a protein band corresponding to the IgD-bindingmolecules were cut out from the gel and solubilized by electronicelution. At reelectrophoresis the purified protein, protein D (D),migrated as a single band (42 kilodaltons) (FIG. 5) without discerniblebreakdown products.

To confirm that protein D was not identical with the previouslydescribed outer membrane proteins 1 or 2 with molecular weights of 49and 39 kilodaltons, respectively, debris (d) and supernatants (s) afterSarcosyl extraction of whole H. influenzae bacteria were subjected toSDS-PAGE, transferred to Immobilon filters and blotted with antibodiesto protein 1 and protein 2 and also with human IgD. As can be seen inFIG. 5 protein D migrates differently from protein 1 and protein 2.

Binding Properties of Protein D

The interaction of protein D with human IgD was further verified in gelfiltration experiments where ¹²⁵I-protein-D was eluted together with IgDwhen a mixture of the two proteins was run on a Sephadex G-200 column(FIG. 6 c). Protein D run alone on the same column was eluted slightlyafter the 43 kilodaltons standard protein (Ovalbumin) confirming theapparent molecular weight of 42 kilodaltons for protein D.

Radiolabeled protein D was also studied in different dot blotexperiments to further examine the binding specificity of the molecule.FIG. 7 shows that protein D effectively bound two highly purified humanIgD myeloma proteins. A distinct reaction could be detected at 0.15 and0.3 μg of the two IgD proteins, respectively. Two additional IgD myelomaproteins which were tested with the same technique could also distinctlybe detected at 0.3 μg (data not shown). In dot blots IgD-Fab fragmentsand IgD-Fe fragments bound protein D at 2.5 and 1.2 μg, respectively. Incontrast 8 different IgG myeloma proteins representing all subclassesand L-chain types showed no visible reaction with protein D at 5 μg.Neither could any reaction between protein D and three monoclonal IgM,one monoclonal IgA preparation, polyclonal IgE or some additionalproteins be detected. However, with polyclonal IgG a weak reaction wasdetected at 5 μg (FIG. 7).

Cloning of the Protein D Gene

DNA isolated from H. influenzae 772 was partially digested with Sau3Aand enriched for fragments in the size of 2 to 7 kilobasepairs (kbp) byfractionation on a sucrose gradient. These fragments were ligated to theBamHI-cut and phosphatase-treated vector pUCI8. E. coli. JM83 cellstransformed with the ligation mixture by high voltage electroporationwere plated selecting for resistance to ampicillin. Individual colonieswere transferred to nitrocellulose filters and screened with a cocktailof monoclonal antibodies (MAbs) as described in Materials and Methods.

Among the 15,000 colonies tested, 60 were found positive. Eight positivecolonies were picked, purified and subjected to another two rounds ofscreening. All clones remained positive during the purification. Thepurified clones were tested for IgD binding with human IgD, rabbitanti-human IgD and peroxidase conjugated goat anti-rabbit Ig:s in acolony immunoassay as described in Materials and Methods. All werepositive regarding IgD binding. Additionally, the clones were foundpositive when screening with the three MAbs individually.

Restriction enzyme analysis of plasmid DNA from the positive clonesshowed that all but one clone carried a 3.3 kbp insert with two internalSau3A sites. One clone contained an additional 2.0 kbp Sau3A fragment.One of the smaller recombinant plasmids, pHIJ32, was chosen for furthercharacterization. A partial restriction enzyme map was established forthe insert of H. influenzae DNA in pHIJ32 (FIG. 8). To identify theregion coding for protein D, restriction enzyme fragments were subclonedinto pUC18. The resulting transformants were tested for expression ofprotein D using colony immunoblot analysis as described above. Theseexperiments showed that plasmids carrying a 1.9 kbp HindIII-ClaIfragment from one end of the insert allowed expression of IgD-bindingprotein. This recombinant plasmid, called pHIC348, was kept for furtherexperiments. The protein D gene cloned in pHIC348 is expressed from apromoter in pUC18. This was shown by cloning the HindIII-ClaI fragmentof pHIJ32 in the opposite orientation in pUC19. All transformantsexpressed IgD binding, as would be expected if the gene is under thecontrol of an endogenous promoter. Transformants carrying theHindIII-ClaI fragment in the opposite direction to pHIC348 grew poorlyand autolysed during cultivation. This was probably due to the lacZpromoter of pUCI9 being oriented in the same direction as the promoterof protein D which led to an overexpression of protein D which waslethal to the cells. In pHIC348 the lacZ promoter was in the oppositedirection of the protein D promoter.

DNA Sequence Analysis of the Protein D Gene

The nucleotide sequence of both strands of the insert from pHIC348 wasdetermined either by direct plasmid sequencing of subclones and deletionconstructs or by subcloning restriction fragments into phages M13mp18and M13mp 19. Commercially available universal and reverse M13 primerswere used. Sequencing was done across all restriction enzyme sites usedin subcloning and the sequencing strategy is outlined in FIG. 8.

The DNA sequence (FIG. 9, SEQ ID NO: 1) reveals an open reading frame of1092 by starting with an ATG codon at position 204 and finishing atposition 1296 with a TAA stop codon. The open reading frame correspondsto a protein of 364 amino acid residues (SEQ ID NO: 3). Ten nucleotidesupstream of the methionine codon is a sequence, AAGGAG, that iscomplementary to the 3′ end of the 16S rRNA of E. coli (Shine, J. andDalgarno, L. Proc. Natl. Acad. Sci. USA, 71:1342, 1974). The spacingbetween the centre of this putative ribosome binding site (rbs) and thestart codon is 13 by in comparison to the average spacing of 10 bp in E.coli. The 5′ flanking region, upstream of the proposed rbs, shows thepresence of possible promoters. The sequences of the −10 region, TAAAAT(151-156), and the −35 region, TTGCTT (127-132), show homology to theconsensus of E. coli promoters (Rosenberg, M. and Court, P., Annu. Rev.Genet, 13:319, 1979) and are identical with promoters recognized by theE. coli RNA polymerase. The spacing between the putative −10 and −35sequences is 18 bp, which is comparable with the favoured value of 17bp.

Between position 1341 and 1359 there is an inverted repeat with thepotential to form a stem and loop structure. This repeat does not,however, resemble a typical rho-independent transcription terminator.

Protein D Structure

The gene for protein D encodes for a protein of 364 amino acid residuesdeduced from the nucleotide sequence (FIG. 9, SEQ ID NO: 3). TheN-terminal amino acid sequence has typical characteristics of abacterial lipoprotein signal peptide (Vlasuk et al., J. Biol. Chem.258:7141, 1983) with its stretch of hydrophilic and basic amino acids atthe N-terminus followed by a hydrophobic region of 13 residues, and witha glycin in the hydrophobic core. The putative signal peptide ends witha consensus sequence Leu-Ala-Gly-Cys (SEQ ID NO: 2), recognized by theenzyme signal peptidase II (SpaseII). The primary translation producthas a deduced molecular weight of 41,821 daltons. Cleavage by SpaseIIwould result in a protein of 346 amino acids with a calculated molecularsize of 40,068 daltons, in contrast to the estimated size of the matureprotein D of approximately 42 kilodaltons. Posttranslationalmodifications of the preprotein may account for this discrepancy.Several attempts to determine the amino-terminal amino acid sequence ofprotein D were performed by applying about 1000 pmoles thereof in anautomated amino acid sequencer. Since no amino acid phenylthiohydantoinderivatives were obtained, the amino-terminal end of the singleIgD-receptor polypeptide chain is probably blocked.

Protein D expressed in E. coli JM83 carrying pHIC348 was analysed inimmunoblotting experiments (FIG. 10). Cytoplasmic, periplasmic andmembrane fractions from cells in late logarithmic phase were separatedon a SDS-PAGE gel and electroblotted to an Immobilon filter. A proteinthat binds all three anti-protein D monoclonal antibodies (16C10, 20G6and 19B4) and radiolabeled IgD could be detected in all three fractions(lane 2-4) from E. coli JM83/pHIC348 as a single band with an estimatedmolecular weight of 42 kilodaltons, i.e. equal or similar to protein Dprepared from H. influenzae (lane 1, FIG. 10).

The nucleotide sequence and the deduced amino ac-id sequence of H.influenzae 772 protein D were compared with other proteins of knownsequence to determine homology by using a computer search in the EMBLand Genbank Data Libraries. Apart from similarities in the signalsequence no homology was found.

SUMMARY

A novel surface exposed protein of H. influenzae or related Haemophilusspecies is described. The protein named protein D is an Ig receptor forhuman IgD and has a apparent molecular weight of 42,000. Protein D canbe detected in all of 116 encapsulated and non-encapsulated isolates ofH. influenzae studied. The protein from all strains shows in addition tothe same apparent molecular weight immunogenic similarities sinceprotein D from all strains interacts with three different mousemonoclonal antibodies and monoclonal human IgD. A method forpurification of protein D is described. Cloning of the protein D genefrom H. influenzae in E. coli is described as well as the nucleotidesequence and the deduced amino acid sequence corresponding to amolecular weight of 41,821 daltons including a putative signal sequenceof 18 amino acids containing a consensus sequence, Leu-Ala-Gly-Lys (SEQID NO: 2) for bacterial lipoproteins.

1-16. (canceled)
 17. A hybridoma cell capable of producing a monoclonalantibody to an immunogenic portion of a surface exposed protein ofHaemophilus influenzae or related Haemophilus species, said proteinhaving an apparent molecular weight of 42,000 and a capacity of bindinghuman IgD.
 18. A purified antibody which is specific to an immunogenicportion of a surface exposed protein of Haemophilus influenzae orrelated Haemophilus species, said protein having an apparent molecularweight of 42,000 and a capacity of binding human IgD.
 19. A method ofdetecting the presence of Haemophilus influenzae or related Haemophilusspecies in a sample by contacting said sample with the antibody of claim18 in the presence of an indicator. 20-22. (canceled)